Mtdna extractor ct kit

De-identified remnant benign margins of resected liver from patients undergoing surgery for hepatic tumours were used as an abundant source of genomic DNA (gDNA) and mtDNA under an IRB-approved protocol. Mitochondrial DAMPs were isolated as previously described.^{32 (link)} Briefly, 200 mg of liver was sonicated on ice at 100% amplitude (30-sec each time with 30-s intervals, 10 times). The disrupted mitochondrial suspensions were centrifuged at 12,000 g for 10 min and at 10,000 g for 30 min. Supernatants were used in experiments as mtDAMPs; the yield of mtDAMPs is similar to that recovered in vivo in experimental 5% liver injury models. mtDNA was extracted using the mtDNA Extractor CT kit (#291-55301, Wako Chemicals, Richmond, VA, USA) following the manufacturer’s protocol. gDNA was extracted using QIAmp DNA Mini Kit following the manufacturer’s protocol (#51304, Qiagen, Germantown, MD, USA).

Mitochondrial DNA separation and 8-oxodG measurement were performed as described previously [11 (link)]. Briefly, mtDNA was purified by the mtDNA Extractor CT kit according to the recommended protocol (Wako Pure Chemical Co., Japan). The mtDNA extraction procedure was optimized to minimize artificial induction of 8-oxodG by using radical-free phenol, minimizing exposure to oxygen, and by the addition of 1 mM deferoxamine mesylate and 20 mM TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), according to the European Standards Committee on Oxidative DNA Damage [20 (link)]. Purified mtDNA was digested to deoxynucleotides by incubation at 37°C with 5 U nuclease P1 (in 20 μL of 20 mM sodium acetate, 10 mM ZnCl₂, 15% glycerol, pH 4.8) for 30 min and with 1 U of alkaline phosphatase (in 20 μL of 1 M Tris-HCl, pH 8.0) for 1 h. The 8-oxodG and dG concentrations were measured by HPLC with online electrochemical and ultraviolet detection, respectively. After filtering through a 0.22 μm filter membrane (Millipore, Bedford, MA, USA), the reaction mixture was applied to a HPLC system with a symmetry C18 column (Waters Associates, Milford, MA, USA) that was attached to a Coulochem electrochemical detector (ESA, Bedford, MA, USA) to measure 8-oxodG. An HP 1100 series UV detector at 254 nm was used to measure dG. The levels of 8-oxodG are expressed as the molar ratio of 8-oxodG to 10⁵ dG.

Isolation of mitochondria from the renal cortex was performed using mitochondria extraction kits (Thermo Fisher Scientific, Rockford, IL, USA) [9 (link)]. Mitochondrial DNA (mtDNA) was extracted from the renal cortex using the mtDNA Extractor CT kit (Wako Pure Chemical Industries, Osaka, Japan) [9 (link)]. 8-OHdG levels in DNase I-digested mtDNA were determined by ELISA (High-Sensitive 8-OHdG Check, Institute for the Control of Aging, Shizuoka, Japan) [9 (link)].